[go: up one dir, main page]

WO1999050277A1 - Ribozymes a tete de marteau et leurs derives dits circulaires, en epingle a cheveux, circulaires/en epingle a cheveux, en lasso, en epingle a cheveux/lasso - Google Patents

Ribozymes a tete de marteau et leurs derives dits circulaires, en epingle a cheveux, circulaires/en epingle a cheveux, en lasso, en epingle a cheveux/lasso Download PDF

Info

Publication number
WO1999050277A1
WO1999050277A1 PCT/US1999/006770 US9906770W WO9950277A1 WO 1999050277 A1 WO1999050277 A1 WO 1999050277A1 US 9906770 W US9906770 W US 9906770W WO 9950277 A1 WO9950277 A1 WO 9950277A1
Authority
WO
WIPO (PCT)
Prior art keywords
ribozymes
circular
hammerhead ribozyme
haiφin
ribozyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/006770
Other languages
English (en)
Inventor
Duane E. Ruffner
Laixin Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Utah Research Foundation Inc
Original Assignee
University of Utah Research Foundation Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Utah Research Foundation Inc filed Critical University of Utah Research Foundation Inc
Priority to CA002324654A priority Critical patent/CA2324654A1/fr
Priority to EP99915078A priority patent/EP1066312A4/fr
Priority to US09/647,314 priority patent/US6307041B1/en
Publication of WO1999050277A1 publication Critical patent/WO1999050277A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/337—Ribozyme

Definitions

  • This invention relates to ribozymes. More particularly, the invention relates to derivatives of wild type hammerhead ribozymes, termed circular, hai ⁇ in, circular/hai ⁇ in, lariat, and hai ⁇ in/lariat hammerhead ribozymes.
  • Ribozymes are RNA or modified RNA molecules that can cleave themselves or other nucleic acid molecules (usually RNA) in a catalytic fashion, similar to traditional protein enzymes.
  • the hammerhead ribozyme is one of the smallest ribozymes currently known, and therefore is the most studied of catalytic RNAs. It has shown great utility as a research tool and an antisense therapeutic composition (e.g., U.S. Patent No. 5,254,678).
  • hammerhead ribozyme The structure and mechanism of the hammerhead ribozyme have been examined using a broad range of approaches. Recently, crystal structures of the hammerhead have been reported. The crystal structures exhibit a Y-shaped configuration for the hammerhead. In this configuration helices I and II form the adjacent upper arms, while helix in forms the lower leg of the Y. Based on these findings, hammerheads in which helix I and ⁇ are constrained to remain adjacent and roughly parallel, are expected to be catalytically active. See NASAdsson, S.T., Tuschl, T., and Eckstein, F. (1995) RNA 1, 575-83.
  • FIG. 1A Crystallographic evidence (Scott, W.G., Finch, J.T., and Klug, A. (1995) Cell 81, 991-1002; Scott, W.G., Murray, J.B., Arnold, J.R.P., Stoddard, B.L., and Klug, A. (1996) Science 274, 2065-9; Pley, H.W., Flaherty, K.M., and McKay, D.B.
  • ribozymes For use of ribozymes in research and especially in therapeutic treatment of various diseases and conditions by antisense-mediated gene inhibition, it would be advantageous to develop hammerhead ribozyme derivatives that have a higher specific activity, have a reduced requirement for magnesium ions, and are more resistant to nuclease degradation than wild-type hammerhead ribozymes.
  • R 1 and R 2 are oligoribonucleotides of at least 2 bases configured for base pairing with a substrate; L is a spacer; and b is 0 or 1, with the proviso that if b is 0, then R 1 and R 2 are bonded together with a phosphodiester bond, and if b is 1, then L is covalently bonded to R 2 .
  • Another embodiment of the invention comprises a hammerhead ribozyme derivative having a structure represented by: R ⁇ L' R' cugauga ⁇ R ⁇ O -R 4 wherein R 1 and R 2 are oligoribonucleotides of at least 2 bases configured for base pairing with a substrate; R 3 and R 4 are oligonucieotides of at least 2 bases configured for base pairing with each other; L 1 and L 2 are spacers; and b and d are 0 or 1.
  • R 1 and R 2 are oligoribonucleotides of at least 2 bases configured for base pairing with a substrate
  • R 3 and R 4 are oligonucieotides of at least 2 bases configured for base pairing with each other
  • L 1 , L 2 , and L 3 are spacers
  • b, d, and f are 0 or 1, with the proviso that if f is 0, then R 3 and R 4 are bonded together by a phosphodiester bond, and if f is 1, then L 3 is bonded to both R 3 and R 4 .
  • Still another embodiment of the invention comprises a hammerhead ribozyme derivative having a structure represented by:
  • R 1 is an oligoribonucleotide of at least 2 bases configured for base pairing with a substrate
  • R 2 is an oligoribonucleotide of at least 2 bases configured for base pairing with R 4
  • R 4 is an oligonucleotide of at least 2 bases configured for base pairing with R 2
  • L 1 and L 2 are spacers
  • Q is a branching moiety
  • b and d are 0 or 1, with the proviso that is b is 0, then R 1 is covalently bonded to Q, and if b is 1, then L 1 is covalently bonded to Q.
  • Yet another embodiment of the invention comprises a hammerhead ribozyme derivative having a structure represented by: R 3 -(L') b -R 1 -(cugauga)-R 2 -(L 2 ) d -R 4 wherein R 1 is an oligoribonucleotide of at least 2 bases configured for base pairing with a substrate; R 2 is an oligoribonucleotide of at least 2 bases configured for base pairing with both R 3 and R 4 ; R 3 is an oligonucleotide of at least 2 bases configured for base pairing with R 2 ; L 1 and L 2 are spacers; and b and d are 0 or 1.
  • the spacers can be independently selected from the group consisting of ribonucleotides, oligoribonucleotides, -0-CH 2 CH 2 CH 2 0-, -0-(CH 2 CH 2 0) 3 -, -O- (CH 2 CH 2 0) 6 -, -0-(CH 2 CH 2 0) 3 -OP0 3 -0-(CH 2 CH 2 O) 6 -, and -0-(CH 2 CH 2 0) 6 -OP0 3 -0- (CH 2 CH 2 0) 6 -.
  • Yet another embodiment of the invention comprises a hammerhead ribozyme derivative having a structure selected from the group consisting of circular ribozymes, hai ⁇ in ribozymes, circular/hai ⁇ in ribozymes, lariat ribozymes, and hai ⁇ in-lariat ribozymes.
  • a method of using a hammerhead ribozyme having a structure selected from the group consisting of circular ribozymes, hai ⁇ in ribozymes, circular/hai ⁇ in ribozymes, lariat ribozymes, and hai ⁇ in-lariat ribozymes comprises contacting a hammerhead ribozyme substrate with said hammerhead ribozyme under conditions configured for digestion of said substrate.
  • FIG. 1A shows open (inactive) and closed (active) conformations of the hammerhead ribozyme formed between a linear substrate (sub; SEQ LD NO: 1) and a linear ribozyme (Lrbz;
  • FIG. IB shows a ribozyme formed between a linear substrate (sub; SEQ ID NO:l) and a covalently closed circular ribozyme (e.g., SEQ ID NO:2; SEQ ID NO:3); the arrow indicates the cleavage site, and X is a linker.
  • FIG. 1C shows a ribozyme formed between a linear substrate (sub; SEQ ID NO:l) and a covalently closed circular/hai ⁇ in ribozyme (e.g., SEQ ID NO:4-8); the arrow indicates the cleavage site, and X, and X 2 are linkers.
  • FIGS. 2A and 2B show preparation of a circular ribozyme and a circular/hai ⁇ in ribozyme, respectively, from linear precursors pre-Crbz (SEQ ID NO:9) and pre-Hrbz (SEQ ID NO: 10) using T4 RNA ligase.
  • FIG. 3A shows cleavage of a substrate (sub) by linear and circular ribozymes; each lane contains the cleavage reaction for the ribozyme indicated, and pre-CrbzLc indicates the cleavage reaction for the linear precursor to CrbzLc.
  • FIG. 3B shows cleavage reactions for linear contaminants; the ribozymes and substrate, as indicated, were incubated under similar conditions used in the cleavage reactions (lanes marked +). As controls, unreacted CrbzLc and CrbzL27 were loaded directly on the gel in lanes designated (-). The positions of the substrate, ribozymes, and cleavage product are indicated by the arrows. "Linear precursor" indicates the position where the linear precursors migrate on the gel and where the nicked circles would appear.
  • FIGS. 4A and 4B show cleavage reactions of the linear and circular/hai ⁇ in ribozymes against substrates sub and sub2, respectively.
  • FIGS. 5A, 5B, and 5C show possible base-pairing configurations for the complex between (A) the substrate, sub, and the circular/hai ⁇ in ribozyme Hrbz, (B) the substrate, sub, and the circular/hai ⁇ in ribozyme HrbzL3', and (C) the substrate, sub2 (SEQ LD NO: 11), and Hrbz.
  • FIGS. 6A and 6B show linear (A) and circular (B) ribozymes used for kinetic analysis: (A) substrate, sub3 (SEQ ID NO: 12), and linear ribozyme, Lrbz3; (B) substrate, sub3, and circular ribozyme, Crbz3.
  • FIG. 7 shows magnesium ion dependence of cleavage of linear and circular ribozymes:
  • FIGS. 8A-G show: (A) wild-type I/ ⁇ format ribozyme (SEQ ID NO: 13) and substrate (SEQ ID NO: 14); (B) an illustrative circular ribozyme (SEQ LD NO: 13) according to the present invention; (C) an illustrative hai ⁇ in ribozyme (SEQ ID NO: 13) according to the present invention; (D) an illustrative circular/hai ⁇ in ribozyme (SEQ ID NO: 13) according to the present invention; (E) wild-type TJI format ribozyme (SEQ ID NO: 15) and substrate (SEQ ID NO: 16); (F) an illustrative lariat ribozyme according to the present invention; (G) an illustrative hai ⁇ in- lariat/ribozyme (SEQ ID NO: 17 and SEQ ID NO: 18) according to the present invention.
  • SEQ ID NO: 13 wild-type I/ ⁇ format
  • hammerhead ribozyme derivatives Five types are disclosed herein: circular, hai ⁇ in, circular/hai ⁇ in, lariat, and hai ⁇ in/lariat (FIGS. 8B-D, F, G). These configurations of the hammerhead are catalytically active and provide advantages not present in the wild-type hammerhead configurations. Since they can be produced by a variety of methods, including both enzymatic and/or chemical procedures, as well as by fully automated synthesis on a DNA synthesizer, the invention is not specific for any particular method of synthesis.
  • circular, hai ⁇ in, circular/hai ⁇ in, lariat, and hai ⁇ in-lariat hammerhead ribozymes synthesized by any means are included in this invention. This would also include circular, hai ⁇ in, circular/hai ⁇ in, lariat, and hai ⁇ in-lariat hammerhead ribozymes synthesized endogenously within cells using suitable gene expression systems.
  • the hammerhead ribozyme derivatives of the present invention offer many advantages relative to the wild-type hammerhead ribozyme, some of which are as follows. All five configurations are locked in a closed and active conformation. This may be an advantage for the study of structure and mechanism of the hammerhead ribozyme. Some of the configurations have been shown to have the advantage of increased activity and a reduced requirement for a divalent metal ion co-factor relative to the wild-type ribozymes. The increased activity and lower metal requirement offers significant advantage for use of these molecules as therapeutic agents and research tools. These new ribozymes also possess an increased resistance to degradation by nucleases relative to wild-type ribozymes, which also represents a significant advantage.
  • Circular and circular/hai ⁇ in ribozymes were prepared as follows. Ten nanomole of the linear RNA precursor and 3.0 ⁇ mole of MgCl 2 were dissolved in 223 ⁇ l H 2 O. This mixture was placed at 90°C for 3 minutes and 16°C for at least one hour. The following was added to the RNA-containing mixture: 30 ⁇ l of 10X buffer (500 mM Tris-HCl (pH 7.7), 100 mM DTT), 30 ⁇ l of 400 mg/ml PEG 8,000, 7.5 ⁇ l of 20 mM ATP and 10 ⁇ l of 20 units/ ⁇ l T4 RNA ligase.
  • 10X buffer 500 mM Tris-HCl (pH 7.7), 100 mM DTT
  • the crude pellet was resuspended and fractionated in 15% or 20% denaturing polyacrylamide gels.
  • the RNA in the gel was visualized by UV shadowing, and the products were recovered by a crush and soak method.
  • the purified RNA was dissolved in 60 ⁇ l H 2 O, producing stock solutions of 60-70 ⁇ M. The total recovered yields were about 40% for most of the preparations.
  • RNA precursors synthesized on a DNA synthesizer were covalently closed into circles using T4 RNA ligase (FIGS. 2A-B).
  • Seven circular and nine hai ⁇ in ribozyme variants were examined (Tables 1 and 2), all differing in the composition of the linkers at positions X (circular), or X, and X 2 (hai ⁇ in).
  • the preferred product was the desired monomer circles or circular hai ⁇ ins.
  • the ligations produced the desired circularized product in 75% yield. More typically, yields of the desired circles were 90% or greater. Linear dimers and trimers were formed as minor products of ligation.
  • T4 RNA ligase and T4 polynucleotide kinase were from New England Biolabs.
  • Shrimp alkaline phosphatase was from United States Biochemical.
  • All DNA and RNA oligonucieotides were synthesized on an Applied Biosystems 394 automated DNA synthesizer on 1.0 pmole scale using the phosphoramidite method.
  • Phosphoramidites of nucleosides, linker 3, linker 9 and linker 18 were from Glen Research; all other synthesis reagents were from Applied Biosystems.
  • a 5' phosphate was added during synthesis.
  • DNA oligonucieotides were purified by ethanol precipitation.
  • RNA oligonucieotides were purified by 15% or 20% denaturing polyacrylamide gel electrophoresis and recovered from the gel by a crush and soak method.
  • the reactions were stopped by adding 15 ⁇ l of stop mix (50 mM EDTA, 7M urea, 0.02% bromphenol blue, and 0.02% xylene cyanole) and run into 20% denaturing polyacrylamide gels. The gels were analyzed using a Molecular Dynamics Phosphorimager to determine the percentage cleaved.
  • stop mix 50 mM EDTA, 7M urea, 0.02% bromphenol blue, and 0.02% xylene cyanole
  • Circular ribozymes were incubated under similar conditions used in the cleavage reactions. Reaction conditions were as follows. One ⁇ M of ribozyme (containing a trace amount labeled with 32 P) and 4.0 ⁇ M substrate (containing a trace amount labeled with 32 P), alone or together, were incubated in 10 ⁇ l of cleavage buffer (50 mM Tris-HCl (pH 8.0), 20 mM MnCl 2 , 0.1% SDS) at room temperature for
  • CrbzLc which possessed a cytidine residue in place of a linker, exhibited activity slightly better than that of the linker L0 (no linker) to L9 variants. Cytidine is equivalent to L3 in terms of contour length, therefore it was expected that CrbzLc and CrbzL3 would possess similar activities.
  • CrbzL 18, CrbzL27, and CrbzL36 exhibited markedly better cleavage activity (FIG. 3A).
  • CrbzL27 and CrbzL36 exhibited activity comparable to the linear ribozyme, Lrbz.
  • the activity of CrbzLl ⁇ was only moderately reduced relative to CrbzL27 and CrbzL36.
  • the circular ribozymes may become nicked during the purification step or alternatively during the cleavage reaction. This is also an unlikely explanation for the activity of the circular hammerheads. Because of their small size, the circular RNAs are not easily nicked in the experimental conditions, especially since all steps were performed at room temperature or lower. Even so, only a small percentage of nicked molecules are likely to be active, since nicks in the catalytic core or within the first 2 nucleotides in helices I and II should produce inactive ribozymes. It was also checked to see if linear contaminants could be observed in the cleavage reactions. For this, circular ribozymes were incubated under the same conditions used for the cleavage reactions and analyzed by denaturing polyacrylamide gel electrophoresis.
  • linkers (L9 or shorter) exhibited low levels of activity, while the longer L18-containing variants possessed activities approaching that of the linear ribozyme.
  • the results of the L3 variants were interesting (FIG. 4A).
  • the two single L3 variants, HrbzL3 and HrbzL3' exhibited cleavage activities comparable to that of the wild type Hrbz.
  • the double L3 variant, HrbzL3.3 exhibited markedly reduced activity. It is not su ⁇ rising that HrbzL3 and HrbzL3' would possess activities similar to the wild type, since the L3 linker approximated in length the single nucleotide it replaced. It was su ⁇ rising that when both the A and G were replaced by L3 linkers (HrbzL3.3), the activity dropped significantly below that for the wild-type Hrbz.
  • HrbzL3.3 was likely the consequence of the nucleotide composition closing helix IV.
  • the wild type hai ⁇ in ribozyme was designed such that the closing base opposition was a G (X,) and an A (X 2 ) on top of a C-G base pair.
  • This motif was chosen because it is the same motif present at the top of the anticodon stem of yeast tRNAphe. In tRNAphe, this motif produces a non-canonical G-A pairing (propeller twist), which serves to widen the end of the anticodon stem and inhibit further stacking above
  • the G and A nucleotides can also participate in Watson-Crick pairings to helices I and II of the substrate.
  • the cleavage activities of the hai ⁇ in ribozymes were examined against a different substrate, sub2 (SEQ ID NO: 11).
  • Sub2 had the same sequence as the wild-type substrate (sub) except that the 5' and 3' nucleotides were switched (see FIGS. 5 A and C).
  • the observed cleavage activity of sub by Hrbz represents the combined activities of the A and B form complexes.
  • Replacement of G or A with L3 blocks the ability to form the non- canonical G-A pair and this produces a new complex A'.
  • Complex A' is similar to complex A in that it lacks the G-A pairing, but it is dissimilar in that it can only form one of the two additional base-pairs possible for form A (FIGS. 5 A and B).
  • the comparable activities of Hrbz, HrbzL3 and HrbzL3' are likely due to the fact that the complexes with HrbzL3 and HrbzL3' will exist in the A' form.
  • the A' form is expected to be intermediate in activity between the A and B forms.
  • sub2/Hrbz complex likely exists in equilibrium between two forms.
  • the two forms are likely to be an A'-like form, designated A", and a B form (FIG. 5C).
  • the A" form is much like the A form of sub/Hrbz, except that only one of the two additional base-pairs is capable of forming, and it is a non-canonical G-U pair.
  • the additional G-U pair is likely to enhance activity of the A" form over the B form.
  • HrbzL3 For sub2 (FIG. 4B) the minor reduction in activity of HrbzL3, relative to Hrbz, is likely due to the loss of the U-G pairing in the A" complex.
  • the increased activity of HrbzL3' is likely due to the elimination of the G-A pairing, which allows the ribozyme/substrate complex to exist solely in the more active A" form.
  • the activity of HrbzL3.3 was expected to be similar to HrbzL3 since neither ribozyme is capable of forming the G-U base-pair.
  • the mildly enhanced activity of HrbzL3.3 relative to HrbzL3 (and Hrbz) might arise due to a favorable increase in conformational flexibility as a result of the dual L3 linkers.
  • HrbzL9.18 and HrbzL 18.18 can only associate with sub through a total of six base- pairs in helices I and ⁇ . For Lrbz, it can form 8 base-pairs to sub. For HrbzL9.18, HrbzL18.18, and Lrbz, all can associate with sub2 through only 6 base-pairs in helices I and LI. Therefore, it is likely that the activities of HrbzL9.18 and HrbzL18.18 against sub would also exceed that of a suitably matched linear ribozyme.
  • the samples contained 20 nM ribozyme and up to 10 different concentrations of
  • Circular Ribozymes A minimal kinetic analysis has been performed on circular ribozymes possessing either the LI 8 or L27 linkers. To ensure that the results would be reliable, the circular ribozymes were based on a linear ribozyme characterized by Clouet, D.O.B., and Uhlenbeck, O.C. (1996) RNA 2,483-91. This ribozyme was shown to be homogeneous on non-denaturing gels, indicating that it does not exist in alternate inactive conformations. Additionally, it is well behaved kinetically. The linear and circular ribozymes use the same substrate, sub3 (SEQ ID NO: 12), which associates with the ribozyme through five base-pairs in each of helices I and II (FIGS. 6 A and 6B).
  • RNA was concentrated by centrifugation and electrophoresed into 20% denaturing PAGE gels, followed by quantitation using a Molecular Dynamics Phosphorimager. The metal dependence of cleavage for Crbz3L18, Crbz3L27, and Lrbz3 were examined.
  • both circular ribozymes appear to be saturated at approximately the same magnesium ion concentration, their levels of activity at saturation are quite different. This suggests that both circular ribozymes have a reduced dependence on magnesium ion, compared to the linear ribozyme, at least in terms of structure.
  • the difference in maximal activity of the circular ribozymes simply reflects a difference in their catalytic efficiencies. This difference in efficiency may be the result of the LI 8 linker being too short for optimal separation of helices I and II, causing the catalytic core of Crbz3L18 to be less optimally folded relative to Crbz3L27. This is supported by the results with the Crbz linker variants (FIG. 3A).
  • the linkers in covalently closed hammerhead ribozymes are likely to influence activity in a number of ways.
  • the linker can influence activity by determining the distance separating helices I and ⁇ .
  • the low activity of the shorter linker containing circles and hai ⁇ ins may be attributed to a less than optimal separation of helices I and ⁇ .
  • the longer linker containing ribozymes appear to possess a more optimal separation.
  • the separation that the longer linkers allow appears to be greater than that needed. For instance, in the crystal structures, helices I and II are separated by approximately 25 ⁇ .
  • linker lengths of 45 ⁇ (CrbzL36) and 37 ⁇ (HrbzL9.9, includes the diameter of helix IV) appear to be necessary. This might suggest, that in addition to influencing activity by affecting the separation of helices I and ⁇ , the linkers may affect activity in another manner.
  • Induced twist may influence activity in multiple ways. It may perturb the structure of the catalytic core to produce a less active ribozyme. This possibility is supported by the observed increase in activity with increasing linker length for the circular and circular/hai ⁇ in ribozymes.
  • induced twist may influence substrate binding (and/or product release). Support for this is the observed higher Km values for
  • FIGS. 8B-D and 8F-G Illustrative circular, hai ⁇ in, circular/hai ⁇ in, lariat, and hai ⁇ in-lariat configured hammerhead ribozymes are shown in FIGS. 8B-D and 8F-G. Also shown are the corresponding wild type IM (FIG. 8A) and I/m (FIG. 8E) ribozymes. For clarity, only the wild-type ribozymes are shown with their substrate molecules. All five of the new ribozyme configurations have been shown to possess cleavage activity. The following notes relate to FIGS. 8A-G.
  • Helices I, ⁇ , HI, and IV are as indicated.
  • the lengths of the four helices can be varied as needed. There are no upper limits other than functionality on the length of the helices, but 2-3 base-pairs appears to be the practical lower limit.
  • the stems can be closed with a loop. Again there is no upper limit other than functionality on the size of the loops, but loops of 3 to 4 nucleotides seem optimal. Since zero, one, two, or three of the stems, in any combination, can be closed by loops, there is great flexibility in how the hammerhead can be constructed.
  • L represents a linker.
  • the linker can be composed of zero (i.e. direct connection) or 1 or more
  • L can be composed of non-nucleotide linkers of varying length, such as, but not limited to, Spacer Phosphoramidites C3, 9, and 18 (Glenn Research, #10-1913, 10-1909, or 10-1918, respectively).
  • X bonds X 2 , and Z represent linkers. The composition of these linkers is defined by the L linker. X, and X 2 may or may not be the same composition and length.
  • Y is a linker and its composition is defined by the L linker.
  • B is a branched connection.
  • the branch point can be placed at the end of the helix II as illustrated, but also within helix ⁇ . In either case, the branch could be produced using an asymmetric branching phosphoramidite (Clontech #5252, Palo Alto, California).
  • the branch can be introduced using modified nucleotides possessing a pendant functional group on the base or sugar, such as, but not limited to, Carboxy-dT (Glenn Research, #10-1035-90).
  • the pendant functional group is used to couple the linker to form the branched structure.
  • Circularization of the circular, circular/hai ⁇ in, and lariat ribozymes can be achieved in a variety of ways. It can be achieved enzymatically using DNA or RNA ligase, both of which are commercially available from several sources. It can be achieved with any number of chemical methods. Circularization typically would be performed after chemical synthesis of the precursor RNA molecules and could use both chemical and enzymatic means. It is also possible to perform the circularization during automated synthesis on a DNA synthesizer using the dT Nucleotide PS support (Glenn Research, #26-2630).
  • oligonucleotide or “oligoribonucleotide” have no particular intended size limitation, unless a particular size is otherwise stated.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des dérivés du ribozyme à tête de marteau appelés ribozymes circulaires, ribozymes en épingle à cheveux, ribozymes circulaires/en épingle à cheveux, ribozymes en lasso, ribozymes en épingle à cheveux/lasso. Lesdits dérivés du ribozyme à tête de marteau ont une activité catalytique, ainsi qu'une activité spécifique accrue et une exigence réduite d'un cofacteur d'ions métalliques bivalents, par comparaison avec le ribozyme à tête de marteau de type sauvage. Ces dérivés peuvent être utilisés en tant que compositions thérapeutiques antisens.
PCT/US1999/006770 1998-03-28 1999-03-29 Ribozymes a tete de marteau et leurs derives dits circulaires, en epingle a cheveux, circulaires/en epingle a cheveux, en lasso, en epingle a cheveux/lasso Ceased WO1999050277A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002324654A CA2324654A1 (fr) 1998-03-28 1999-03-29 Ribozymes a tete de marteau et leurs derives dits circulaires, en epingle a cheveux, circulaires/en epingle a cheveux, en lasso, en epingle a cheveux/lasso
EP99915078A EP1066312A4 (fr) 1998-03-28 1999-03-29 Ribozymes a tete de marteau et leurs derives dits circulaires, en epingle a cheveux, circulaires/en epingle a cheveux, en lasso, en epingle a cheveux/lasso
US09/647,314 US6307041B1 (en) 1998-03-28 1999-03-29 Circular, hairpin, circular/hairpin, lariat, and hairpin-lariat hammerhead ribozymes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7979198P 1998-03-28 1998-03-28
US60/079,791 1998-03-28

Publications (1)

Publication Number Publication Date
WO1999050277A1 true WO1999050277A1 (fr) 1999-10-07

Family

ID=22152835

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/006770 Ceased WO1999050277A1 (fr) 1998-03-28 1999-03-29 Ribozymes a tete de marteau et leurs derives dits circulaires, en epingle a cheveux, circulaires/en epingle a cheveux, en lasso, en epingle a cheveux/lasso

Country Status (4)

Country Link
US (1) US6307041B1 (fr)
EP (1) EP1066312A4 (fr)
CA (1) CA2324654A1 (fr)
WO (1) WO1999050277A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014375A3 (fr) * 2001-08-09 2003-10-16 Archemix Corp Molecules de sonde d'acide nucleique et methodes d'utilisation de ces dernieres
US6863792B1 (en) 2001-10-11 2005-03-08 The Ohio State University Method of making electrochemical detectors based on iridium oxide
US7125660B2 (en) 2000-09-13 2006-10-24 Archemix Corp. Nucleic acid sensor molecules and methods of using same

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6274369B1 (en) * 1996-02-02 2001-08-14 Invitrogen Corporation Method capable of increasing competency of bacterial cell transformation
JP2003535599A (ja) 2000-06-06 2003-12-02 ティーエム バイオサイエンス コーポレイション 核酸の捕捉部とその使用
CA2374388C (fr) 2002-02-28 2005-07-12 Matthew C. Coffey L'utilisation de ribozymes pour la detection d'agents adventifs
US20040005546A1 (en) * 2002-02-28 2004-01-08 Oncolytics Biotech Inc. Use of ribozymes in the detection of adventitious agents
EP1578767A4 (fr) * 2002-12-04 2008-01-09 Algos Therapeutics Inc Procedes et materiaux de modulation des trpm2

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5652094A (en) 1992-01-31 1997-07-29 University Of Montreal Nucleozymes
US6096880A (en) * 1993-04-15 2000-08-01 University Of Rochester Circular DNA vectors for synthesis of RNA and DNA
CA2295207A1 (fr) * 1997-06-19 1998-12-23 Innovir Laboratories, Inc. Ribozymes en tete de marteau a fonction de clivage etendue

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
PUTTARAJU M., ET AL.: "CIRCULAR RIBOZYMES GENERATED IN ESCHERICHIA COLI USING GROUP I SELF-SPLICING PERMUTED INTRON-EXON SEQUENCES.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 271., no. 42., 18 October 1996 (1996-10-18), US, pages 26081 - 26087., XP002921168, ISSN: 0021-9258, DOI: 10.1074/jbc.271.42.26081 *
See also references of EP1066312A4 *
WANG L., ET AL.: "NOVEL CONFIGURATIONS OF THE HAMMARHEAD RIBOZYME: INCREASED ACTIVITY AND REDUCED MAGNESIUM ION REQUIREMENT.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 250., no. 03., 1 September 1998 (1998-09-01), US, pages 711 - 719., XP002921170, ISSN: 0006-291X, DOI: 10.1006/bbrc.1998.9393 *
WANG L., ET AL.: "OLIGORIBONUCLEOTIDE CIRCULARIZATION BY 'TEMPLATE-MEDIATED' LIGATION WITH T4 RNA LIGASE: SYNTHESIS OF CIRCULAR HAMMERHEAD RIBOZYMES.", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD., GB, vol. 26., no. 10., 12 May 1998 (1998-05-12), GB, pages 2502 - 2504., XP002921169, ISSN: 0305-1048, DOI: 10.1093/nar/26.10.2502 *
WANG L., ET AL.: "RATIONAL DESIGN OF A NEW HAMMERHEAD RIBOZYME CONFIGURATION: LARIAT HAMMERHEAD RIBOZYMES AS IMPROVED ANTISENSE THERAPEUTICS.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, US, vol. 120., no. 31., 1 May 1998 (1998-05-01), US, pages 7684 - 7690., XP002921171, ISSN: 0002-7863, DOI: 10.1021/ja980796q *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7125660B2 (en) 2000-09-13 2006-10-24 Archemix Corp. Nucleic acid sensor molecules and methods of using same
WO2003014375A3 (fr) * 2001-08-09 2003-10-16 Archemix Corp Molecules de sonde d'acide nucleique et methodes d'utilisation de ces dernieres
US6863792B1 (en) 2001-10-11 2005-03-08 The Ohio State University Method of making electrochemical detectors based on iridium oxide

Also Published As

Publication number Publication date
EP1066312A1 (fr) 2001-01-10
EP1066312A4 (fr) 2002-01-02
CA2324654A1 (fr) 1999-10-07
US6307041B1 (en) 2001-10-23

Similar Documents

Publication Publication Date Title
US6890908B1 (en) Modified ribozymes
US5817635A (en) Modified ribozymes
JP7050866B2 (ja) オリゴヌクレオチドの産生のための新規プロセス
Costa et al. Frequent use of the same tertiary motif by self‐folding RNAs.
AU645083B2 (en) Site specific cleavage of single-stranded DNA
EP0291533B1 (fr) Ribozyme d'arn endoribonucleases de restriction et procedes
US5712128A (en) Enzymatic RNA molecules
US5639647A (en) 2'-deoxy-2'alkylnucleotide containing nucleic acid
US9012140B2 (en) Target-dependent nucleic acid adapter
EP0594584A1 (fr) Enzymes d'acide nucleique pour le clivage d'adn
JP2021507699A (ja) オリゴヌクレオチドの産生のための新規プロセス
US20060036087A1 (en) 2'-deoxy-2'-fluoro modified RNA
Doudna et al. Miniribozymes, small derivatives of the sunY intron, are catalytically active
Bartel et al. Template-directed primer extension catalyzed by the Tetrahymena ribozyme
US6307041B1 (en) Circular, hairpin, circular/hairpin, lariat, and hairpin-lariat hammerhead ribozymes
Tsukahara et al. Inhibition of HIV-1 replication by triple-helix-forming phosphorothioate oligonucleotides targeted to the polypurine tract
Wang et al. Novel configurations of the hammerhead ribozyme: Increased activity and reduced magnesium ion requirement
WO2004024864A2 (fr) Enzymes d'adn de clivage de l'arn a regioselectivite ou enantioselectivite modifiee
Bossmann Sabine Müller, Rüdiger Welz, Sergei A. Ivanov, and
Richardson Targeting RNA tertiary structure: Tethered oligonucleotide probes and cleavage-agent modified RNA
Giver It is generally believed that the earliest progenitor of modern life was as et of chemical shapes or reactions that

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2324654

Country of ref document: CA

Ref country code: CA

Ref document number: 2324654

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1999915078

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 09647314

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1999915078

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1999915078

Country of ref document: EP